The maintenance of genomic integrity following DNA damage depends on the coordination of the DNA repair system and cell cycle checkpoint controls. Increasing evidence suggests that several tumor suppressors, including p53 and BRCA1, participate in these processes. Chk2, a DNA damage-activated protein kinase, regulates p53 and BRCA1 activities following gamma radiation. In addition, heterozygous germ-line mutations in Chk2 have been identified in Li-Fraumeni syndrome (LFS), a highly penetrant familial cancer phenotype usually associated with mutations in the p53 gene. The observation that Chk2 is mutated in a sub-group of Li-Fraumeni patients that lack mutations in p53 gene strongly suggests that, like p53, Chk2 is a tumor suppressor gene. We hypothesize that Chk2 participates in a DNA damage-responsive pathway that is critical for the prevention of cancer development. In this proposal, we will explore the molecular mechanisms by which Chk2 functions as a tumor suppressor. 1: We speculate that Chk2 exerts its tumor suppressor function mainly by regulating p53. Our preliminary studies have shown that Chk2 phosphorylates the C-terminus of p53. Here, we will explore the contribution of these phosphorylation events to the regulation of p53 following DNA damage. 2: We will study the roles of forkhead homology-associated (FHA) domain in the regulation of Chk2 functions. Two tumor-associated mutations within Chk2 FHA domain have been identified, suggesting that the FHA domain of Chk2 is important for its tumor suppression function. Studies of its homologue in yeast also suggest that the FHA domain may play essential roles in regulating Chk2 function. Based on the recently solved structure of the yeast Chk2 FHA domain, we will perform structural and functional analyses of the Chk2 FHA domain and assess the roles of the FHA domain in Chk2 activation and p53 stabilization following gamma radiation. 3: Chk2 is a component of a large protein complex with an apparent molecular weight of approximately 200 kDa. Using mass spectrometry and microsequencing analyses, we have demonstrated that one of the Chk2-associated proteins is a novel protein with unknown function. In this proposal, we will clone the cDNA encoding this polypeptide and explore whether its association with Chk2 contributes to Chk2 function. In addition, we have observed that, following gamma radiation, the Chk2-containing protein complex increases in size. We speculate that there are additional proteins associating with Chk2 following DNA damage. We will identify these damage-regulated, Chk2-associated proteins, perform initial characterization of their interactions with Chk2 and begin to explore the biological significance of these damage- regulated interactions. Collectively, these studies will elucidate the Chk2-dependent DNA damage-signaling pathway and the molecular mechanisms by which Chk2 functions as a tumor suppressor.